Discovery of potent small-molecule inhibitors of lipoprotein(a) formation.

Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and apolipoprotein(a) (apo(a))1,2. Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (KIV) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs. 3-7). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) KIV7-8. We identify compounds that bind to apo(a) KIV7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a). Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329-which is already in phase 2 studies-as a potent and specific orally administered agent for reducing the levels of Lp(a).

Comparative genomic and proteomic analysis of cytoskeletal changes in dexamethasone-treated trabecular meshwork cells.

Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included ß3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit α, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and ß3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.

Dexamethasone-associated cross-linked actin network formation in human trabecular meshwork cells involves ß3 integrin signaling.

PURPOSE: To determine whether cross-linked actin networks (CLANs) formed in dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells are structurally similar to those formed after ß3 integrin activation and involve αvß3 integrin signaling. METHODS: Two HTM cell strains and an αvß3 integrin-overexpressing immortalized TM cell line were used. DEX- or ethanol-pretreated HTM cells were plated on fibronectin with or without ß3 integrin-activating mAb AP-5. Immunofluorescence microscopy was used to identify phalloidin-labeled CLANs and to ascertain the presence of α-actinin, PIP(2), and syndecan-4 within them. ß3 Integrin signaling involvement was determined using a PI3-kinase (LY294002) or Rac1 (NSC23766) inhibitor. αvß3 Integrin expression levels and the ß3 integrin activation state were determined by fluorescence-activated cell sorter analysis and immunofluorescence microscopy. RESULTS: CLANs associated with either DEX treatment or ß3 integrin activation contained syndecan-4, PIP(2), and α-actinin. In the absence of mAb AP-5, LY294002 did not affect DEX-associated CLAN formation, whereas NSC23766 decreased the percentage of CLAN-positive cells by 80%. In the presence of mAb AP-5, both inhibitors decreased DEX-associated CLAN formation. DEX pretreatment increased ß3 integrin-induced CLAN formation nearly sixfold and the level of αvß3 integrin expression and activation threefold compared with control cells. Activated ß3 integrin-positive adhesions increased nearly fivefold in DEX-treated cells. αvß3 Integrin overexpression in TM-1 cells increased CLAN formation twofold. CONCLUSIONS: DEX-associated CLANs were structurally similar to those induced by mAb AP-5 and involved both increased expression and activation of αvß3 integrins. Thus, glucocorticoid-induced CLAN formation may involve enhanced ß3 integrin signaling in HTM cells, possibly by an inside-out signaling mechanism.

Identification of heme as the ligand for the orphan nuclear receptors REV-ERBalpha and REV-ERBbeta.

The nuclear receptors REV-ERBalpha (encoded by NR1D1) and REV-ERBbeta (NR1D2) have remained orphans owing to the lack of identified physiological ligands. Here we show that heme is a physiological ligand of both receptors. Heme associates with the ligand-binding domains of the REV-ERB receptors with a 1:1 stoichiometry and enhances the thermal stability of the proteins. Results from experiments of heme depletion in mammalian cells indicate that heme binding to REV-ERB causes the recruitment of the co-repressor NCoR, leading to repression of target genes including BMAL1 (official symbol ARNTL), an essential component of the circadian oscillator. Heme extends the known types of ligands used by the human nuclear receptor family beyond the endocrine hormones and dietary lipids described so far. Our results further indicate that heme regulation of REV-ERBs may link the control of metabolism and the mammalian clock.